IN THE UNITED STATES PATENT OFFICE
`
`In re Application of:
`
`Calderari, et al.
`
`Serial No.: 11/186,311
`
`Filing Date:
`
`July 21,2005
`
`Title: LIQUID PHARMACEUTICAL
`FORMULATIONS OF
`PALONOSETRON
`
`)
`)
`)
`)
`)
`)
`)
`)
`)
`)
`)
`
`DECLARATION OF DANIELE BONADEO, M. Chern. Pharm.
`37 C.F.R. § 1.132
`
`4)
`
`5)
`
`I, Daniele Bonadeo, hereby give this declaration.
`My name is Daniele Bonadeo.
`1)
`I am an inventor for this application.
`2)
`I am also Director, Head of Corporate Technical Affairs, Manufacturing Operations, for
`3)
`Helsinn Healthcare SA ("Helsinn"), the assignee of the above-referenced patent
`app!ication.
`One of my job responsibilities at Helsinn is to ensure that Helsinn's drug products are
`stable, and that adequate testing is performed to ensure such stability.
`One of Helsinn' s main products is palonosetron hydrochloride, which is marketed in the
`United States as Aloxi®. The product is marketed in injectable form and marketing
`authorization has been obtained for gel-cap dosage form for the treatment of nausea and
`vomiting from chemotherapy, radiotherapy and general surgery.
`Numerous stability studies have been performed on the injectable formulation of
`to the formulation and
`to evaluate how changes
`palonosetron over the years
`manufacturing process would impact stability. These studies were performed by Helsinn,
`our contract manufacturers, and the owner of palonosetron before we acquired the drug.
`
`6)
`
`2536648vl
`
`Helsinn Healthcare Exhibit 2072
`Dr. Reddy's Laboratories, Ltd., et al. v. Helsinn Healthcare S.A.
`Trial PGR2016-00008
`
`Page 1 of 9
`
`

`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. 111186,311
`Page 2 of9
`All of our studies, regardless of the formulation parameter which was varied, show that
`the stability of palonosetron generally improves as the concentration of palonosetron is
`reduced, and that concentration is the most important determinant of product stability.
`Table 1 contains the results of one of our stability studies, conducted in a phosphate-
`buffered, saline solution at pH 7.4.
`
`7)
`
`8)
`
`TABLE 1. Palonosetron HCI Concentration-Stability Study (pH 7.4, 40 °C)
`'% Palonosetron HCI Remaining at
`Palonosetron HCI Cone.
`(mg/ml, as free base)
`8weeks
`100
`101
`99
`23
`49
`
`1 week
`
`2 weeks
`
`5weeks
`
`-
`
`-·
`
`100
`
`99
`102
`
`~
`
`100
`101
`93
`73
`
`57
`
`0.01
`0.1
`1.0
`10
`50
`
`9)
`
`As can be seen, the stability of the molecule improves in this formulation as its
`concentration decreased, with greatest stability seen below 0.1 mg/ml. We made this
`same observation in other studies, as discussed in greater detail below.
`10) We also performed a pH-stability study to determine the best pH at which to formulate
`the molecule. The study was conducted with 60 mcg/ml palonosetron aqueous solutions,
`buffered at pH 2.0, 5.0, 7.4 and 10.0. No ingredients were present other than the pH
`adjusting agent, pH buffer, and palonosetron. The results are reported in Table 2.
`
`TABLE 2. Palonosetron HCI 80 °C pH-Stability Study
`Buffer
`pH at Reaction
`Temp.
`2.0
`5.0
`
`pHatRoom
`t----· Temp.
`2.0
`5.0
`
`0_01 M HCT
`Acetate
`
`T9o
`(days)_
`76
`Not determined. 99.2%
`remaining at 252 days
`180
`270
`
`7.4
`10
`
`7.3
`9.4
`
`Phos2hate
`Carbonate
`
`I
`
`2536648vl
`
`Page 2 of 9
`
`

`
`132 Declaration ofDaniele Bonadeo
`U.S.S.N. 11/186,311
`Page 3 of9
`
`11)
`
`13)
`
`The results demonstrate that the molecule is extremely stable at a pH of 5.0, when
`maintained at a low palonosetron concentration such 60 mcg/ml, and that stabilizers and
`the like are unnecessary to maintain that stability.
`12) We also conducted additional studies to evaluate the impact of various excipients on
`stability, and improve the stability even further.
`After settling on mannitol and a citrate buffer for the fonnulation for practical reasons,
`we studied the effect of palonosetron and EDTA concentration on stability, maintaining
`the pH constant at approximately 5.0, and keeping the same tonicifying agent (mannitol)
`and buffering agent (trisodium citrate). Stability was measured based on the percentage
`of palonosetron that remained undegraded at I, 2, 3 and 6 months, under standard
`conditions of accelerated stability testing (i.e. 40 °C).
`The results are reported below in Table 3.
`
`14)
`
`TABLE 3. Formulation Optimization Study
`
`2S36648vl
`
`Page 3 of 9
`
`

`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. 11/186,311
`Page4 of9
`
`Degradation Products
`{Total % Lab~I Strength}
`Storage Time (mo.) at 40 C
`
`Formulation (1)
`
`Pal(}.Conc.
`
`{2)
`0.10
`
`0.10
`
`0.40
`0.40
`0.40
`
`0.40
`
`0.40
`
`1.00
`
`1.00
`1.20
`1.20
`2.50
`
`Buff
`er
`Con
`c.
`(3)
`2.0
`
`EDTA
`Cone.
`
`(%w/v}
`0.000
`
`40
`
`60
`10
`35
`
`10
`
`60
`
`20
`
`20
`40
`20
`80
`
`0.000
`
`0.000
`0.000
`0.050
`
`0.100
`
`0.100
`
`0.050
`
`0.050
`0.000
`0.000
`0.000
`
`Potency
`{%label Strength)
`Storage Time (mo.) at 40C
`
`1
`103
`
`106
`
`99
`98
`103
`
`102
`
`99
`
`2
`99
`
`103
`
`98
`97
`103
`
`101
`
`99
`
`102
`
`102
`
`100
`99
`99
`
`99
`96
`97
`93
`
`3
`101
`
`106
`
`100
`97
`103
`
`101
`
`100
`
`100
`
`99
`97
`98
`91
`
`6
`100
`
`104
`
`95
`95
`102
`
`100
`99
`99
`98
`8B
`95
`
`Rate
`Const.
`
`(4)
`-0.29
`
`-0.61
`
`-0.75
`-0.59
`-0.15
`
`·0.29
`
`-0.27
`
`-0.35
`
`-0.31
`-1.73
`-0.55
`-3.25
`
`0
`
`1
`0.30
`
`0.24
`
`0.07
`0.13
`0.07
`
`0.13
`
`0.00
`
`0.21
`
`0.15
`0.36
`0.03
`
`2
`0.6B
`
`0.49
`
`0.14
`0.09
`0.06
`
`0.17
`
`0.00
`
`0.08
`
`0.07
`1.00
`0.37
`4.04
`
`--
`
`-
`3
`0.61
`
`0.46
`
`0.46
`0.22
`0.21
`
`0.06
`
`0.02
`
`0.28
`o.;w
`1.59
`0.61
`7.81
`
`Rat
`e
`Con
`st."_
`(4)
`0.15
`
`0.11
`
`0.33
`0.12
`0.03
`
`6
`0.97
`
`0.70
`1.92
`0.71
`0.16
`
`0.10
`
`0.01
`
`0.13
`
`0.02
`
`0.37
`
`0.22
`8.77
`1.73
`
`0.06
`
`0.03
`1.49
`0.30
`2.52
`
`1.45
`
`0
`10
`1
`10
`9
`99
`99
`10
`2
`10
`1
`10
`1
`10
`0
`99
`97
`98
`10
`1
`10
`0
`10
`0
`10
`0
`10
`0
`99
`10
`0
`10
`1
`99
`10
`1
`10
`0
`99
`98
`
`2:.50
`
`2.50
`
`2.50
`
`2.70
`
`2.70
`5.00
`
`5.00
`
`5.00
`5.00
`
`5.00
`
`5.00
`5.00
`
`40
`
`20
`
`80
`
`35
`
`10
`60
`
`10
`
`0
`60
`
`60
`60
`10
`
`0.000
`
`0.000
`
`0.050
`
`0.000
`
`0.050
`0.000
`
`0.000
`
`0.000
`0.050
`
`0.050
`
`0.100
`0.100
`
`91
`
`96
`
`97
`
`97
`
`99
`
`102
`
`99
`98
`
`100
`
`98
`98
`
`97
`
`93
`95
`
`94
`
`95
`
`97
`
`99
`
`100
`97
`99
`93
`94
`
`96
`
`91
`85
`
`100
`
`98
`100
`
`102
`
`99
`99
`
`101
`
`98
`98
`
`-1.75
`
`-1.50
`
`-1.11
`
`-1.58
`
`-0.48
`-1.16
`
`·.075
`
`-1.92
`-2.41
`
`-1.66
`
`-2.82
`·4.37
`
`2.77
`
`2.65
`
`0.79
`
`2.72
`
`1.22.
`2.90
`
`2.02
`
`3.95
`3.56
`
`4.19
`
`4.29
`4.23
`
`4.39
`
`4.04
`
`1.07
`
`2.76
`
`1.17
`5.56
`
`2.86
`
`7.83
`8.14
`
`7.10
`
`7.55
`13.87
`
`1.22
`
`1.40
`2.25
`
`1.24
`
`1.93
`3.10
`
`2.86
`
`2.65
`2.19
`
`1.34
`
`0.36
`
`7.21
`
`1.17
`
`3.03
`
`0.43
`
`1.
`
`2.
`3.
`4.
`
`15)
`
`Lots are sorted by Palonosetron HCI concentration, EDTA concentration and buffer concentration.
`All lots contain mannitol as the tonicifying agent.
`Palonosetron concentrations are in mglmL free base equivalents.
`Citrate buffer concentrations are millimolar. All formulations are pH 5. 0.
`Rate constants are calculated with degradation product concentrations in two significant digits.
`
`One notable observation from these results is that the presence of EDT A improves
`stability at low palonosetron concentrations, but actually decreases stability at high
`pa!onosetron HCl concentrations.
`
`2536648vl
`
`Page 4 of 9
`
`

`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. 1l/l86,311
`Page 5 of9
`
`16)
`
`17)
`
`18)
`
`The fact that EDTA improves the stability ofpa!onosetron at all is somewhat surprising,
`given our earliest wmk with the molecule, in which palonosetron demonstrated
`comparable stability at 5 oc as it did at 60-1 00 ac. If the molecule were undergoing
`auto-oxidation (the typical reason for adding a chelating agent), one would expect the
`initiators and a faster reaction and
`higher temperature to produce more radical
`degradation.
`The fact that the chelating agent consistently improves the molecule's stability only at
`lower concentrations is also an intriguing discovery.
`Based on the results in Table 3, we prepared several graphs to inform our analysis,
`including the response surface plot depicted below as Figure 1. This graph plots
`degradation rate constant as a function of EDTA concentration and palonosetron HCl
`concentration, holding the buffer strength constant at 20 mM. A lower degradation rate
`constant indicates a more stable fonnulation.
`
`FIGURE 1
`
`19)
`
`As can be seen, the stability of palonosetron improved as its concentration was reduced
`across the entire range of conditions studied. This same result can be seen when the
`buffer strength is varied, as shown below in Table 4. These results are consistent with the
`I, and reinforce our opinion that palonosetron
`results reported above in Table
`concentration is a critical factor for the stability of palonosetron fonnulations.
`
`253664Svl
`
`Page 5 of 9
`
`

`
`EDTACone.
`(%w/w)
`
`TABLE4
`palonosetron
`Citrate Cone.
`Cone. (mg/ml)
`(mM)
`0.4
`5
`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. 11/186,311
`Page 6 of9
`
`% Degt·adation
`at6 mo.
`0.71%
`2.86%
`
`0.97%
`1.73%
`4.04% (3 mo:)_
`
`0.7%
`8.77%
`4.39% (3 mo.)
`
`0.1%
`13.87 (3 mo.)
`
`0.13%
`7.55% (3 mo.)
`
`0.1
`1.2
`2.5
`
`0.1
`1.2
`2.5
`
`0.4
`5
`
`0.4
`5
`
`0
`0
`
`0
`0
`0
`
`0
`0
`0
`
`0.1
`0.1
`
`0.1
`O.l
`
`10
`10
`~ -~ ---
`20
`20
`20
`
`-~v--~-
`
`40
`40
`40
`
`10
`10
`
`60
`60
`
`20) We also prepared a contour plot of potency rate constant as a function of buffer and
`EDT A concentrations based on the data repmied in Figure 3, at a low palonosetron
`concentration (0.4 mg/ml). This plot is depicted below in Figure 2:
`
`20
`
`-40
`nuiff<>r'
`
`60
`
`eo
`
`21)
`
`FIGURE2
`As can be seen from this figure, at the low palonosetron concentration depicted, there is a
`region of no apparent degradation with EDT A from 0.025 to 0.075 % w/v and buffer
`from 10 to 40 mM. This region is marked by the e:J symbol.
`
`2536648vl
`
`Page 6 of 9
`
`

`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. ll/186,311
`Page 7 of9
`
`intravenous
`the stability of the
`The foregoing results should be contrasted with
`formulation reported in example 13 of the Berger 5,202,333 patent ("Berger '333").
`than we
`'333 formulation used a higher palonosetron concentration
`The Berger
`eventually settled upon (10-l 00 mg/ml for the Berger '333 formulation vs. 0.05 mg/ml
`for the optimum formulation in the present invention), and a lower pH (3. 7 for the Berger
`'333 formulation vs. 5.0 for the optimum formulation in the present invention).
`The following Table 5 compares the formulations described in Berger '333 example 13
`and example 4 ofthe current application, and their relative stability:
`TABLES
`Berger '333 Example 13
`Intravenous Formulation
`10-100 mg *
`
`Example 4 of Present
`Application
`0.05 mg
`
`··~
`
`Palonosetron
`Hydrochloride
`pH
`Dextrose monohydrate
`tonicifying agent
`Mannitol tonicifying agent
`Citric acid monohydrate
`Sodium hydroxide**
`EDTA
`Trisodium citrate
`Water fo~ injection
`
`3.7
`q.s. to render isotonic
`
`5.0
`--
`41.5 mg/ml
`1.56 mg
`q.s. to pH 5.0 ± 0.5
`0.5mg
`3.7mg
`g.s. to l.O ml
`
`--
`1.05 mg
`0.18 mg
`--
`--
`q.s. to 1.0 ml
`YES
`NO
`1-2 Year Stabili!Y?
`*Asswning compound offormula I in Example 13 is palonosetron
`**Assumes formulation requires pH increase
`This comparison shows, once again, that palonosetron concentTation and pH have the
`most significant impact on the stability of the formulation.
`
`22)
`
`23)
`
`24)
`
`25)
`
`2516648vl
`
`Page 7 of 9
`
`

`
`132 Declaration of Daniele Bonadeo
`U.S.S.N. 11/186,31 I
`Page 8 of9
`
`26)
`
`The formulations differ in terms of tonlcifying agent, but 1 am unaware of any literature
`that would suggest that changing the tonicifYing agent from dextrose to mannitol in a
`system such as this can have a bearing on the stability of the formulation. A study we
`performed comparing mannitol and saline, summarized below in table 6, supports this
`opinion.
`TABLE 6. Chemical Stability Summary ofPalonosetron HCI Injection Citrate
`Formulations
`
`Formulation (1)
`
`Tonicifier
`
`I
`
`Lot#
`
`I.
`
`l
`
`[
`Degradation
`Potency
`! Products (Total %
`(%Label Strength)
`Label Strength)
`AtO mo. At 1 mo. AtO mo. At 1 mo.
`Buffer
`40°C
`40°C
`40°C
`40°C
`Con e.g)
`1.85
`0.46
`96
`95
`20
`l Mannitol
`18635-DML-045-05
`4.32
`1.41
`93
`94
`40
`18635-DML-045-04 Mannitol
`3.80
`94
`95
`1.20
`80
`1863 5-DML-04 5-06 Mannitol
`2.18
`96
`0.46
`98
`Saline
`20
`18635-DML-045-08
`4.54
`0.44
`98
`94
`40
`Saline
`18635-DML-045-07
`3.10
`1.50
`96
`98
`80
`Saline
`18635-DML-045-09
`Palonosetron concentration is 10 rng/mL as free base equivalents. Lots do not contain
`EDT A.
`Citrate buffer concentrations are millimolar. All formulations are pH 5.0.
`
`27)
`
`2.
`The formulations also differ in terms of the buffering mechanism (Berger '333 uses citric
`acid as a buffering agent while Example 4 uses citric acid and sodium citrate). However,
`the buffering agent is incorporated to maintain the solution at the pt·escribed pH, and
`would not be expected to significantly influence the stability of the formulation except
`through its pH maintenance capacity.
`
`2536648vl
`
`Page 8 of 9
`
`

`
`132 Declaration ofDaniele Bonadeo
`U.S.S.N.Il/186,311
`Page 9 of9
`
`I declare that all statements made herein of my own knowledge are true and that al! statements
`made on information and belief are believed to be true, further, that these statements were made
`with the knowledge that willful false statements and the like so made are punishable by fine or
`imprisonment, or both, under Section 1001 of Title 18 of the United States Code. I declare under
`penalty ofpet:jury that the foregoing is true and correct.
`
`Dated: February m_, 2009
`
`Daniele Bomideo
`
`2536648vl
`
`Page 9 of 9